PROJECT SUMMARY Human rhinovirus (RV) is the major pathogen of common cold, asthma, and asthma exacerbation. Airway epithelial cells have been the focus for RV research as it is believed to be the primary site for infection and replication. On the other hand, inflammatory cells including monocytes and macrophages are abundant in diseased airways. Although both epithelial cells and monocytes/macrophages play important roles in RV- induced pathogenesis, their interactions are rarely studied. It is a long-held belief that RV can enter monocytes/macrophages and induce proinflammatory cytokine expression in the absence of viral replication. In our recent publication, we demonstrated for the first time that a major-group RV entered, replicated and induced significant cytokine production in monocytic cell line (THP-1) in the presence of airway epithelial cells or their conditioned media (CM). In our preliminary data, we further showed that primary macrophages from bronchoalveolar lavage (BAL) were also able to support RV replication in the presence of CM. These data suggest a significant role of epithelial cells in regulating permissiveness of monocytes/macrophages for RV infection. Interestingly, the only two previously documented events of monocytic RV replication occurred in THP-1 that was differentiated to macrophage by a PMA protocol, but they failed in finding any underlying mechanism. In our study, increased ICAM1 was found to be both necessary and sufficient to drive RV16 replication in THP-1 treated by CM or by the same PMA protocol, suggesting the involvement of a shared pathway between the two treatments. But, our CM model is obviously more physiologically relevant than the PMA model. To identify the potential epithelial factor(s) that drive this change, we performed size fractionation, in-solution digestion, liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis. 4 protein candidates were discovered. Thus, protein(s) in CM likely drive the change of permissiveness of monocytic cells. Therefore, we hypothesize that epithelial factor(s) renders monocytes/macrophages permissive for RV replication via an ICAM1 mediated mechanism, thereby exacerbating inflammation and potentially leading to RV-associated airway illnesses. We will address this hypothesis in two aims. Specific Aim1 will determine the molecular basis of induced permissiveness of monocytes/macrophages for RV infection by testing two related working hypotheses: 1) increased ICAM1 enhances viral entry and cellular viral burden, thereby circumventing antiviral defense; and/or 2) increased ICAM1 weakens cellular antiviral defense. Specific Aim2 will identify and characterize soluble factor(s) in airway epithelium CM that induces the change of permissiveness for RV infection. The success of this project will have significant impact on the development of therapeutics for treating RV-induced airway illnesses by elucidating novel targets (i.e. permissiveness-inducing factor and replicating-RV-bearing monocytes/macrophages).